Project Summary Malaria remains one of the most important infectious diseases in the world. A fully effective vaccine would be a huge addition to our armamentarium. The most promising vaccine candidate to date, RTS,S, a subunit vaccine composed of a portion of the sporozoite's major surface protein, CSP, has shown limited efficacy that wanes significantly after the first year. Though this falls short of community established goals, RTS,S provides a substrate upon which we can build to create a more efficacious vaccine. The studies outlined in this proposal aim to elucidate the interactions between host and parasite at the inoculation site with the goal of improving future vaccine design. Sporozoites are inoculated into the skin of the mammalian host as mosquitoes search for blood. Few sporozoites are injected, making this a bottleneck for the parasite. After their inoculation, sporozoites are actively motile in the skin, and must find and penetrate blood vessels to enter the circulation. Our studies demonstrate that the majority of sporozoites take 20 to 120 minutes to exit the dermis and only a small proportion succeed in entering the blood circulation. Once in the circulation, sporozoites go to the liver and enter hepatocytes within minutes. Sporozoite passage through the dermis is an understudied phase of malaria infection: Yet, the low numbers of inoculated sporozoites together with the discovery that the parasite is extracellular for the longest period of time in the skin, suggest that this is a time of extreme vulnerability. My laboratory has been studying the dynamics of sporozoite transmission for several years and we are now using quantitative intravital imaging to better understand the requirements for successful exit from the dermis. In Aim 1, we will perform intravital imaging studies with the human malaria parasite Plasmodium falciparum, quantitatively analyzing its motility and blood vessel interactions, in both mouse skin and human skin xenografts. Comparative analysis with rodent malaria sporozoites will identify conserved and species- specific aspects of dermal exit and define metrics that predict successful exit from the dermis. These studies will also enable the development of an in vivo platform to screen vaccine candidates with P. falciparum sporozoites. Previous studies in mice and in RTS,S immunized humans have shown that CSP-specific antibodies correlate with protection, however, the location and mechanism(s) by which these antibodies impact sporozoites are not known. In Aims 2 and 3, we will perform intravital imaging and infection studies with rodent and human malaria sporozoites to determine: a) whether antibodies targeting the two leading sporozoite vaccine candidates have their its greatest impact in the skin; b) the degree to which functional antibodies rely on their ability to inhibit sporozoite motility versus their ability to opsonize sporozoites and direct their destruction by innate immune cells and c) whether fast gliding and cell traversal enable the sporozoite, at least in some cases, to escape the innate host response and the inhibitory activity of antibody. We feel confident that the knowledge gained from these studies will inform the design of future malaria vaccine candidates.